Use of na+ channel blockers and aspirin in manufacturing drugs for producing analgesia synergistically in mammals

ABSTRACT

This invention relates to the use of combinations of a sodium channel blocking compound that binds to an SSI or SS2 site of extracellular region of a sodium channel alpha subunit, and aspirin in manufacturing drugs for producing synergistically analgesic effect in mammals. Pharmaceutical compositions based upon this invention can enhance analgesic effect and reduce dosage of aspirin, therefore side effects and adverse reactions are decreased accordingly.

[0001] This invention relates to the use of combinations of a sodiumchannel blocking compound that binds to an SSI or SS2 site ofextracellular region of a sodium channel alpha subunit, and aspirin inmanufacturing drugs for producing synergistically analgesic effect inmammals. Pharmaceutical compositions based upon this invention canenhance analgesic effect and reduce dosage of aspirin, therefore sideeffects and adverse reactions are decreased accordingly.

[0002] Pharmacologically, anti-inflammation drugs consist of two majorkinds: aspirins and steroids. Aspirin is a very widely used non-steroidanalgesic, as well as an anti-inflammatory analgesic. Belonging to thecategory of acetylsalicylic acids, aspirins mainly compriseacetylsalicylic acids (commonly known as aspirin), salicylates (mainlysodium salicylates) and diflunisall. Salicylic acid is the activeingredient in a salicylate.

[0003] Inhibition of prostaglandin (PG) synthesis is the major mechanismof action for aspirin-alike drugs to produce pharmacological,therapeutic, and toxic and side effects. Aspirin has such effects asinhibiting synthesis of pain sensation exciting substances likebradykinin and histamine, restraining activity of white blood cells,influencing the body temperature adjusting center in the hypothalamus,thereby producing analgesic, anti-inflammatory and antipyretic effects.Aspirin also impairs thromboxane (TXAT) synthesis by inhibitingprostaglandin cyclooxygenase in platelets, thereby inhibiting plateletaggregation. (Xiaozhi CHENG, Pingtian XIAO, Zhongshen WANG, New Editionof Practical Manual for Drugs, 1994, SSI0034400, Chaoxing DigitalLibrary).

[0004] Aspirin has a remarkable analgesic effect in alleviating paincaused by common cold, as well as treating headache and fever induced bygeneral mental stress. It is used mainly for treatment of the followingindications:

[0005] 1. Common cold, fever, mild to moderate pain (headache, dentalpain, neuromuscular pain, menstrual pain et cetera.);

[0006] 2. Rheumatism, rheumatic joint arthritis;

[0007] 3. Generation of thrombus. Regimen of small doses for long termis necessary.

[0008] Aspirin may cause side effects as following (Qingwei SUN, Yi HOU,Novel Clinical Uses of Aspirin-Alike Drugs and Adverse Effects, 1998,SS10034347, Chaoxing Digital Library):

[0009] 1. Stomach pain, occasionally gastric ulcer and bleeding; asthma,skin rash

[0010] in allergic reactions; occasionally reversible hepatic or renaldamage.

[0011] 2. Overdose reactions: mild reactions include salicylism; severeones

[0012] comprise hematuria, convulsion, hallucination, psychiatricdisorder, and

[0013] difficulty in respiration.

[0014] 3. Long term use of aspirin is associated with false positiveresults in examination of sugar in urine, false escalation in serum uricacid, abnormal level of transaminase, decrease in cholesterol,hypokalemia,

[0015] and prolonged thrombinogenesis.

[0016] In 1990s, aspirin was found to have statistically significanteffect on preventing stroke and heart diseases in middle-aged people ifit was taken frequently. Apparently, aspirin possesses such mildanti-coagulation property that it prevents blood clots, therebyimproving blood circulation.

[0017] Aspirin is inexpensive while delivering sound therapeutic effectswith minor adverse reactions, so it is widely used as anover-the-counter drug. However, aspirin at large doses at someoccasions, particularly when needed to produce desirable suchtherapeutic effects as alleviating refractive pain induced by rheumatismand arthritis, could cause gastric ulcer, ischemia, or bleeding in theupper gastrointestinal tract. Although the bleeding is not of bigamount, it will become a serious problem if aspirin is taken at largedoses for a continuous period. Especially when there has been ailment inthe gastrointestinal tracts, overdose of aspirin could even cause death,or at least intoxication symptoms like ulcer, gastric dilatation, andthinned anterior gastric branches. In U.S. Pat. No. 4,491,574, Seifer etal provided in 1985 a solution for diminishing intoxication of aspirinby taking vitamin A simultaneously or in advance so as to increasegastric secretion. This invention discloses an alternative approach,which is to reduce dosage of aspirin by combining a synergisticanalgesic in case large doses of aspirin are required for producinganalgesia.

[0018] Tetrodotoxin (TTX) is a potent non-protein neurotoxin possessingpharmacological effects like analgesia, local anesthesia andanti-convulsion. TTX noticeably alleviates various types of dull painand sharp pain, and does not induce dependence. However, its value forclinical application is limited by dosage. From the perspective ofpracticality, the synergistic interaction between drugs is studied. Inorder to measure the probability of using TTX as a synergistic analgesicclinically, a chemical stimulation model, namely acetic acid inducedwrithing test in mice (sensitive to antipyretic analgesics) was employedto observe the interaction between small doses of TTX and aspirin, anantipyretic and analgesic drug.

[0019] The mechanism of action for TTX to produce analgesia is toinhibit the generation and transmission of neuron pulse by blocking theTTX-sensitive (TTX-S) sodium channels thus the inward sodium current.Aspirin as an antipyretic and analgesic drug inhibits cyclooxygenase soas to impair synthesis of prostaglandin (PG) and to depress thepain-inducing and hyperalgesic effect of prostaglandin (PGE2), andalleviate bradykinin's pain-inducing effect as well.

[0020] It has been disclosed in literature (Cesare P, Mcnaughton P,Peripheral pain mechanisms. Curr Opin Neurobiol 7(4):493-9, 1997 Aug)that hyperalgesia caused by tissue injury is related to two mechanismsat the least: sodium current induced by bradykinin which increasesthermal irritation, and gate voltages of several types ofPGE2-influenced ion channels including TTX-resistant sodium channels.The hyperalgesia effect of PGE2 is related to TTX-R sodium channels(Khasar S G; Gold M S; Levine J D. A tetrodotoxin-resistant sodiumcurrent mediates inflammatory pain in the rat. Neurosci Lett,256(1):17-20, 1998 Oct 30), as PGE2 raises the amplitude ofTTX-resistant sodium current (TTX-RINa), thereby enhances the activityof TTX-R sodium channel. Under the circumstances of chronic pain, thesensitization of nociceptors are mediated through TTX-R sodium channels(Tanaka M; Cumnmins T R: Ishikawa K; Dib-Hajj S D; Black J A; WaxmanSQSNS Na+ channel expression increases in dorsal root ganglion neuronsin the carrageenan in flammatory pain model. Neuroreport,9(6):967-721998 Apr 20), (Krai M G; Xiong Z; Study RE ,Alteration of Na+ currentsin dorsal root ganglion neurons from rats with a painful neuropathy.Pain 81(1-2): 15-24 1999 May). Therefore, analgesia effect can beproduced by blocking TTX-R sodium channels (Akopian A N; Souslova V;England S; Okuse K; Ogata N; Ure J; Smith A; Kerr B J; McMahon S B;Boyee S; Hill R; Stanfa L C; Dickenson A H; Wood J N. Thetetrodotoxin-resistant sodium channel SNS has a specialized function inpain pathways. Nat Neurosci, 2(6):541-8 1999 Jun). This explains thatthe analgesic effect of TTX did not increase markedly with escalatingdoses in a previous heat-induced tail flick test in mice. Aspirininduces analgesia by impairing synthesis of PGE2 and thus decreasing thesodium current conveyed by TTX-R channels. Therefore, synergisticanalgesia by combining aspirin and TTX is hypothesized as they mayjointly inhibit TTX-sensitive and TTX-resistant sodium channelssimultaneously.

[0021] The acetic acid-induced writhing in mice indicated thattetrodotoxin yielded 40.6% and 27.7% inhibition at doses of {fraction(1/25)} and {fraction (1/50)} LD50 (0.79 μg/kg, 0.39 μg/kg),respectively, which was in accordance with the literature (Data andReferences of Main Pharmacodynamics Studies for Tetrodotoxin Injection,Drug Product File 12). When combined with aspirin, tetrodotoxin at thetwo dose levels reduced the half inhibition dose (ID50) of aspirin from44.1 mg/kg alone to 5.0 mg/kg, 10.0 mg/kg, and the 95% inhibition dose(ID95) from 361.8 mg/kg alone to 94.5 mg/kg, 154.3 mg/kg, respectively.Isobolographic analysis proved that there was significant synergisticinteraction between aspirin and TTX.

[0022] This invention is meaningful in that it provides a novel approachfor treating pain clinically, particularly some types of acute andchronic pain which do not respond well to current antipyreticanalgesics, by using small doses of tetrodotoxin in combination so as toimprove analgesic effect and reduce dosage of involved drugs, therebyreducing adverse reactions.

EXAMPLE

[0023] 1 Materials and Methods

[0024] 1.1 Animals

[0025] Kunming mice, 18-22 grams, half male and half female, supplied bythe Experimental Animal Center of Beijing University, Medical Branch.Quality Certificate No. 013056. Classification: One.

[0026] 1.2 Test Article and Reagents

[0027] Tetrodotoxin (TTX), 95% purity, supplied by Nanning Maple LeafPharmaceutical Co., LTD., batch no. 0324C. Diluted with citric acidbuffer solution to required concentration. Aspirin (ASP), powder, 99%purity, manufactured by Shandong Xinhua Pharmaceutical Factory, batchno. 0005564. Ground and then diluted with 0.5% sodium carboxymethylcellulose (CMC) solution. Glacial acetic acid, analytical pure,manufactured by Beijing 52952 Chemical Factory, batch no. 991117.

[0028] 1.3 Methods

[0029] Acetic acid-induced writhing test in mice (Shuyun XU, RulianBIAN, Xiu CHEN, Methodology of Pharmacology Experiments). 380 mice wereselected, given no food but drinking water 12 hours prior to theexperiment, randomly divided into 19 groups: control group (CMCsolution), solely ASP groups (25 mg/kg, 50 mg/kg, 1OO mg/kg, 150 mg/kg,200 mg/kg, totally five groups), solely TTX groups ({fraction (1/25)}and {fraction (1/50)} LD50 doses, or 0.79, 0.39 ug/kg, respectively),and combined groups: TTX (0.39 ug/kg) +ASP(6 mg/kg, 12.5 mg/kg, 25mg/kg, 50 mg/kg, 75 mg/kg), TTX (0.79 ug/kg)+ASP(3 mg/kg ,6 mg/kg, 12.5mg/kg,25 mg/kg,50 mg/kg,75 mg/kg). Solely TTX or ASP was given to miceintramuscularly. For combined groups, drugs were given to both sides ofa mouse intramuscularly at a volume of 0.1 mL/10 g, respectively. After40 minutes, 0.6% glacial acetic acid solution was givenintraperitoneally to induce pain. In the following 15 minutes, writhingmovements were observed and recorded. Sign of a writhing movement wasrecognized to be positive when a mouse manifested repeated contractionof lumbar muscle, inward contraction of stomach, stretch of trunk andhind limbs, upward movement of buttock. The writhing inhibition rate wascalculated according to the following formula:

Inhibition rate (%)=(the writhing incidences in the control group−thoseof a test group)/the writhing incidences in the control group×100%

[0030] The half inhibition rate (IDso) was determined by the probitmethod.

[0031] 1.4 Statistical Analysis

[0032] The SPSS software was employed for the statistical analysis,while the isobolographic analysis was performed to test the drug-druginteraction. (Duanzheng XU, Application of Biostatistics inPharmacology, Science Publishing, 1986, 357-359), (Shuqin YANG, MedicalStatistics, Encyclopedia of Chinese Medical Sciences, Shanghai Scienceand Technology Publishing, 1985, 197).

[0033] 2. Results

[0034] As shown in Table 1, aspirin alone had a half inhibition dose(ID50) of 44.11 mg/kg in the acetic acid-induced writhing assay in mice.Combined with small doses of TTX ({fraction (1/25)} and {fraction(1/50)} LD50), aspirin had its ID50 reduced to 5.01 mg/kg and 96 mg/kg,respectively, and ID95 reduced to 94.47 mg/kg, 154.33 mg/kg from 361.77mg/kg, respectively. The reduction of both ID50 and ID95 exceeded twofolds. TABLE 1 Synergistic Action between TTX and Aspirin (i.m.) byMouse Writhing Assay Average ID₅₀ and 95% Doses No. of WrithingInhibition Confidence Interval ID₉₅ and 95% Confidence Groups (mg/kg)animals movements Rate (%) (mg/kg) Interval (mg/kg) Sodium carboxymethyl50 20 39.0 ± 15.4 — cellulose solution, control TTX 0.79 × 10⁻³ 20 23.2± 11.7 40.6 0.39 × 10⁻³ 20 28.2 ± 9.65 27.7 ASP 25 20 25.1 ± 14.5 35.650 20 19.3 ± 13.8 50.6 44.1 (24.9˜61.9) 361.8 (197.3˜1689.2) 100 20 12.0± 9.2  69.3 150 20 5.2 ± 5.7 86.6 200 20 2.7 ± 1.9 93.2 TTX(0.79μg/kg) + ASP 3.0 20 23.0 ± 8.3  40.9 6.0 20 20.3 ± 12.2 47.9  5.0(3.8˜6.3)  94.5 (62.7˜170.7) 12.5 20 9.5 ± 9.0 75.7 25.0 20 7.2 ± 6.981.6 50.0 20 4.0 ± 4.6 89.9 75.0 20 1.8 ± 1.2 95.4 TTX(0.39 μg/kg) + ASP6.0 20 25.0 ± 10.9 35.8 12.5 20 17.0 ± 8.9  56.4 10.0 (7.6˜12.3) 154.3(99.8˜301.1) 25.0 20 10.0 ± 11.1 74.5 50.0 20 5.8 ± 5.3 85.2 75.0 20 4.5± 3.3 88.6

1.-10. (canceled)
 11. A composition comprising at least one sodiumchannel blocking compound that binds to a SS1 or SS2 site of theextracellular region of a sodium channel α subunit and at least onecompound that is a cyclooxygenase inhibitor.
 12. The composition ofclaim 11, in which the cyclooxygenase inhibitor is salicylic acid or aderivative or salt thereof.
 13. The composition of claim 11, wherein theamount of the at least one sodium channel blocking compound is effectiveto provide a synergistic effect to the at least one cyclooxygenaseinhibitor in producing analgesia.
 14. The composition of claim 11, inwhich the sodium channel blocking compound is at least one selected fromthe group consisting of tetrodotoxin, a derivative of tetrodotoxin,saxitoxin, a derivative of saxitoxin and mixtures thereof.
 15. Thecomposition of claim 11, in which the at least one sodium channelblocking compound is a saxitoxin having a molecular formula C₁₀H₁₇N₇O₄.16. The composition of claim 11, in which the at least one sodiumchannel blocking compound is selected from the group consisting oftetrodotoxin, dehydrotetrodotoxin, aminotetrodotoxin,methoxytetrodotoxin, ethoxytetrodotoxin, deoxytetrodotoxin, tetrodonicacid and mixtures thereof.
 17. The composition of claim 11, in which atleast one cyclooxygenase inhibitor is at least one selected from thegroup consisting of acetylsalicylic acid, sodium salicylate anddiflunisal.
 18. The composition of claim 13, in which the at least onesalicylic acid or derivative or salt thereof is selected from the groupconsisting of acetylsalicylic acid, sodium salicylate and diflunisal.19. The composition of claim 14, in which the at least one salicylicacid or derivative or salt thereof is selected from the group consistingof acetylsalicylic acid, sodium salicylate and diflunisal.
 20. Thecomposition of claim 16, in which the at least one salicylic acid orderivative or salt thereof is acetylsalicylic acid.
 21. The compositionof claim 20, in which the sodium channel blocking compound istetrodotoxin.
 22. The composition of claim 13 that provides a dosage offrom 0.01 to 20 μg of the at least one sodium channel blocking compoundper kilogram body weight of the subject.
 23. The composition of claim 13that provides a dosage of from 0.02 mg to 200 mg of the at least onesalicylic acid or a derivative or salt thereof per kilogram body weightof the subject.
 24. A method for producing analgesia in a subjectcomprising administering to the subject an analgesically effectiveamount of the composition of claim
 11. 25. The method for producinganalgesia in a subject comprising administering to the subject ananalgesically effective amount of the composition of claim
 13. 26. Themethod of claim 25, in which at least one cyclooxygenase inhibitor issalicylic acid or a derivative or salt thereof.
 27. The method of claim25, in which the sodium channel blocking compound is at least oneselected from the group consisting of tetrodotoxin, a derivative oftetrodotoxin, saxitoxin, a derivative of saxitoxin and mixtures thereof.28. The method of claim 26, in which at least one salicylic acid orderivative thereof is selected from the group consisting ofacetylsalicylic acid, sodium salicylate and diflunisal.
 29. The methodof claim 28, in which the sodium channel blocking compound istetrodotoxin.
 30. The method of claim 24, in which the composition isadministered by injection.
 31. The method of claim 30, in which theinjection is an intramuscular injection.
 32. A method for producinganalgesia in a subject comprising administering to the subject an amountof at least one cyclooxygenase inhibitor effective to inhibit synthesisof prostaglandin in said subject and an amount of at least one sodiumchannel blocking compound that binds to a SS1 or SS2 site of theextracellular region of a sodium channel a subunit that provides forsynergistic analgesic effect of the at least one cyclooxygenaseinhibitor.
 33. The method of claim 32, in which at least onecyclooxygenase inhibitor is a salicylic acid or a derivative or saltthereof.
 34. The method of claim 32, in which the at least onecyclooxygenase inhibitor is administered in a dosage form separate fromthe at least one sodium channel blocking compound.
 35. The method ofclaim 33 in which the at least one cyclooxygenase inhibitor isadministered in a dosage form separate from the sodium channel blockingcompound.
 36. The method of claim 32 in which the at least onecyclooxygenase inhibitor is administered in a dosage form together withthe at least one sodium channel blocking compound.
 37. The method ofclaim 32, in which the sodium channel blocking compound is at least oneselected from the group consisting of tetrodotoxin, a derivative oftetrodotoxin, saxitoxin, a derivative of saxitoxin and mixtures thereof.38. The method of claim 33, in which at least one cyclooxygenaseinhibitor is salicylic acid or derivative thereof is selected from thegroup consisting of acetylsalicylic acid, sodium salicylate anddiflunisal.
 39. The method of claim 38, in which the sodium channelblocking compound is selected from the group consisting of tetrodotoxin,a derivative of tetrodotoxin, saxitoxin, a derivative of saxitoxin andmixtures thereof.